Choosing an Enrichment Method for Targeted Sequencing

The advantages of enriching a next-generation sequencing (NGS) library for your genomic regions of interest are many—costs are lower, for example, and the deeper coverage is better able to detect rare mutations or variants. But choosing a target enrichment method first requires considering important factors that may influence your outcome. “The best place to start is with the biological question rather than the technology itself,” says Brittany Niccum, Senior Commercial Product Manager at Integrated DNA Technologies (IDT). “Ultimately, there is no universal best enrichment method; the optimal choice depends on balancing experimental objectives.” This article discusses guidance for choosing between probe-based hybridization capture or PCR-based amplification methods.

PCR-based methods

There are advantages and disadvantages to each method. “The hybridization-based method works well with large [gene] panels and whole-exome sequencing but is more expensive, whereas the amplicon-based method is less expensive but only suited for a smaller number of gene targets,” says Boshen Gao, Senior Applications Scientist at Roche Diagnostics. “Other factors, such as workflow complexity, turnaround time, and automation capability, are also worth considering.”

The multiplex PCR-based amplification method is often a more straightforward protocol with an easier workflow. Here, target enrichment is combined with the NGS library preparation step prior to sequencing, resulting in fewer overall protocol steps compared to the hybrid capture method. Basically, primers designed to flank the region of interest are used in PCR reactions to amplify the target sequence, with multiplex PCR used to amplify multiple targets simultaneously. Another advantage of the PCR method is that it requires so little starting material, only 10 to 100 ng DNA, compared to the hybrid capture method, which may require at least a microgram. This is an especially important consideration when using rare, precious, or degraded samples.

Often used for target enrichment of only a few genes (i.e., up to around 50), the PCR-based amplicon method generally involves smaller target regions or gene panels. “Amplicon-based methods are frequently chosen for smaller, well-defined targets because they can offer streamlined workflows, deep coverage, and cost-efficient sequencing,” says Niccum. “We also offer amplicon-based enrichment through xGen™ Amplicon and rhAMPseq™ CRISPR Analysis System technologies, which leverage multiplex PCR for efficient sequencing of focused target regions and applications such as pathogen sequencing, genotyping, and CRISPR editing analysis.”

The hybrid capture method

In the hybrid capture method, biotinylated oligonucleotide probes hybridize to your target sequences and “capture” your DNA of interest. “You immobilize the molecules on magnetic beads, then wash away everything else, so you end up with this reduced representation library that should be highly enriched for the target region that you’re interested in, and so that’s what you sequence,” says Fiona Kaper, VP of Assay R&D at Illumina. This method underlies Illumina’s main target enrichment tools, though they also offer PCR amplicon-based sequencing as well.

The hybrid capture method is appropriate for most sample types as long as the DNA isn’t too degraded. “The hybridization-based capture method generally requires higher quality and quantity of DNA/RNA input, but specific workflows can be optimized for limited or degraded samples, such as FFPE tissue, if sufficient input mass is available,” says Gao. Roche offers KAPA HyperCap tools for hybridization-based target enrichment.

In contrast to the PCR method, the hybrid capture method is generally used for relatively larger genomic regions or panels. “Hybridization capture is often preferred for larger panels (greater than 50,000 bp), exomes, discovery-focused studies, and applications where broader genomic coverage is important,” says Niccum. IDT’s xGen NGS platform supports hybrid capture in applications including exome sequencing, oncology research, inherited disease, and infectious disease research.

Most hybrid capture protocols employ single-stranded DNA probes, but those from Twist Bioscience use double-stranded probes. “This improves capture uniformity and reduces duplicate reads compared to single-stranded probes,” says Emily Leproust, CEO and Co-founder of Twist Bioscience. “In practice, this means more usable data per run, which matters most in applications like liquid biopsy and minimal residual disease monitoring, where every read counts toward detecting a rare variant at low allele fraction.”

A potential advantage of the hybrid capture method is that probes can pull down library fragments despite the presence of single nucleotide polymorphisms (SNPs) or mutations within them. In contrast, a SNP or mutation underlying a PCR primer, “especially if it’s near the end of a primer, could have a big impact on the efficiency with which you amplify that particular fragment,” says Kaper. “If you use a probe-based hybrid capture method for your enrichment, it’s much more tolerant of underlying SNPs or mutations.”

The hybrid capture method also allows for flexibility in future growth for research projects. “Hybrid capture is particularly valuable when researchers need to cover broader regions, add new targets over time, or support applications that may expand as the research evolves,” says Leproust. “Because it is less constrained by the primer interactions that can arise in highly multiplexed [PCR-based] assays, hybrid capture can scale more readily from targeted panels to much larger designs and whole exomes.” Examples include gene panels for rare diseases, or cancer profiling panels—both of which benefit from regularly updating as new biomarkers are discovered.

In addition

Variations or extensions of these target enrichment methods continue to produce new tools. For example, Roche offers KAPA HyperPETE for target enrichment of smaller panels (< 250 kb), RNA fusions, and ultra-low DNA inputs such as cell-free DNA samples isolated from blood. It uses primer extension-based enrichment (PETE), a hybrid-capture variant that offers a simpler protocol than standard PCR-based enrichment.

In addition, IDT’s Archer™ portfolio uses the Anchored Multiplex PCR (AMP™) method, which detects and enriches for gene fusions. “For applications such as fusion detection, specialized approaches like AMP may provide advantages because they can identify rearrangements without requiring prior knowledge of both fusion partners, while also supporting targeted analysis of DNA variants,” says Niccum. Ultimately, the choice of enrichment method for targeting sequencing will depend on the nature of the project, and which method provides more distinct advantages.